Name: reconstitution of septin assembly at membranes to study biophysical properties and functions
Uploaded: 2022-07-28T14:45:00
Description: Watch this Scientific Journal Video about Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions at JoVE.com

这项工作得到了美国国立卫生研究院（NIH）拨款号的支持。R01 GM-130934和美国国家科学基金会（NSF）授予MCB-2016022。B.N.C.、E.J.D.V.和K.S.C.部分得到了国家普通医学科学研究所在T32 GM119999奖项下的资助。

注意：形成支撑的脂质双分子层需要制备单分散的小单层囊泡（SUV）。请参阅之前发布的关于SUV形成的协议24 。简而言之，所有SUV都是通过探针超声处理形成的，总共70%的幅度为12分钟，通过4分钟的超声处理周期，然后在冰水中休息2分钟。SUV解决方案必须明确，尺寸必须单分散。SUV的尺寸分布可以例如通过动态光散射25来测量。
1. 平?...

<ol>
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细胞膜具有许多不同的形状、曲率和物理化学性质。为了研究细胞构建微米级组件的纳米级机械，有必要设计膜模拟物的最小重构系统。该协议提供了精确控制膜曲率和成分的技术，同时允许用户使用广泛使用的显微镜技术轻松进行定量荧光测量。
该协议的最关键组成部分是在适当的表面上组装孔并处理脂质。这里描述的孔是使用PCR管和UV活化粘合剂手工制作的;重要的是，?...

细胞的形状及其许多内部隔室取决于它们周围的脂质膜。膜是粘弹性结构，可以通过与蛋白质的相互作用，脂质分选以及作用的内部和外部力来产生各种形状而变形1，2，3，4。这些形状通常用膜曲率来描述。细胞使用一套多样化的蛋白质，能够优先组装或“感知”特定的膜曲率，以确保对包括细胞运输，细胞分裂和迁移在内的过程进行定义的时空控制5，6。由于难以平衡时间和空间分辨率与细胞健康，因此很难观察到膜上细胞机制的动力学。虽然超分辨率技术可以提供这些结构的详细视图，但它们需要长时间的采集，而这些采集不适合大多数机械的装配/拆卸时间尺度。此外，这些组件在其天然环境中的分子复杂性以及单个组分可以发挥的众多作用使得最小重构系统成为研究分子功能能力的宝贵工具。
已经开发了最小膜模拟物来研究细胞外的膜特性和蛋白质 - 膜相互作用。膜模拟物从独立的脂质双分子层（例如脂质体或巨型单层囊泡）到支撑脂质双分子层（SLBs）7，8，9，10不等。SLB是锚定在下层支撑物上的仿生膜，通常由玻璃，云母或二氧化硅11，12组成。可以使用各种几何形状，包括平面，球体，棒，甚至起伏或微图案底物，以同时探测凹曲率和凸曲率上的蛋白质 - 膜相互作用1 3，14，15，16，17，18.双层形成始于囊泡吸附到亲水表面上，然后熔合和破裂以形成连续的双层（图1）19。支撑的双层特别适合光学和电子显微镜检查，提供比细胞中通常可以实现的更好的时间和空间分辨率。弯曲的SLB特别提供了一种有吸引力的方法，可以在没有显着的膜变形的情况下探测蛋白质曲率灵敏度，使人们能够区分曲率传感和曲率感应，这在独立系统中通常是不可能分离的。
Septins是一类形成细丝的细胞骨架蛋白，以其在正弯曲膜上组装的能力而闻名6，18，20。在酵母细胞周期的过程中，隔膜蛋白组装成一个环，并且必须重新排列以形成与芽出现和细胞分裂相关的沙漏和双环结构，分别为21。虽然已经使用铂复制电子显微镜在不同的细胞周期阶段22观察隔膜蛋白结构，但使用酵母中的光学显微镜观察隔膜蛋白随时间的组装已经遇到了有限的空间分辨率。以前使用透射电子显微镜（TEM）可视化的脂质单层的隔膜蛋白的工作能够重建几个有趣的隔膜结构，如环，束和纱布23。然而，与荧光显微镜不同，EM技术在时间分辨率方面同样受到限制。为了更好地解决隔膜组装的多尺度过程的动力学参数，我们转向了支持的膜模拟物，在那里人们可以仔细控制膜的几何形状，样品条件和成像模态。
这里描述的方案使用平面或弯曲的SLB，纯化的蛋白质和显微镜技术的组合。定量荧光共聚焦显微镜和全内反射荧光显微镜（TIRFM）用于测量本体蛋白质与各种膜曲率的结合，以及测量单分子的结合动力学。此外，该方案已被调整为与扫描电子显微镜（SEM）一起使用，以检查不同膜曲率上的蛋白质超微结构。虽然这些方案的重点是隔膜细胞骨架，但可以很容易地修改实验方案，以研究读者感兴趣的任何蛋白质的曲率敏感性。此外，那些在内吞作用或水泡运输等领域工作的人可能会发现这些技术可用于探测多蛋白复合物的曲率依赖性组装体。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL PCR Tubes with flat cap, Natural</td>
			<td>Watson</td>
			<td>137-211C(EX)</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL low adhesion tubes</td>
			<td>USA Scientific</td>
			<td>1405-2600</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta mercaptoethanol (BME)</td>
			<td>Sigma-Aldrich</td>
			<td>M6250-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A4612-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverglass for making PEGylated coverslips</td>
			<td>Thermo Scientific</td>
			<td>152450</td>
			<td>Richard-Allan Scientific SLIP-RITE Cover Glass 24x50 #1.5</td>
		</tr>
		<tr>
			<td>DOPC</td>
			<td>Avanti Polar Lipids</td>
			<td>850375</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg Liss Rhodamine PE</td>
			<td>Avanti Polar Lipids</td>
			<td>810146</td>
			<td></td>
		</tr>
		<tr>
			<td>EMS Glutaraldehyde Aqueous 25%, EM Grade</td>
			<td>VWR</td>
			<td>16220</td>
			<td></td>
		</tr>
		<tr>
			<td>EMS Sodium Cacodylate Buffer</td>
			<td>VWR</td>
			<td>11652</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 200 proof</td>
			<td>Fisher Scientific</td>
			<td>04-355-223EA</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane</td>
			<td>Sigma-Aldrich</td>
			<td>440191</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>VWR</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl cellulose 4000cp</td>
			<td>Sigma-Aldrich</td>
			<td>M052-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Microglass coverslips for planar bilayers</td>
			<td>Matsunami</td>
			<td>Discontinued</td>
			<td>22x22</td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Functionalized Silica Microspheres</td>
			<td>Bangs Laboratories, Inc.</td>
			<td>Depends on size: SS0200*-SS0500*</td>
			<td>Silica in aqueous suspension</td>
		</tr>
		<tr>
			<td>Optical Adhesive</td>
			<td>Norland Thorlabs</td>
			<td>NOA 68</td>
			<td>Flexible adhesive for glass or plastics</td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Millipore Sigma</td>
			<td>20816-12-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>VWR</td>
			<td>52858-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Plasma Etch</td>
			<td>PE-25</td>
			<td>Voltage: 120V, 60Hz. Current: 15 AMPS</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>VWR</td>
			<td>0395-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Round coverglass, #1.5 12mm&#160;&#160;</td>
			<td>VWR</td>
			<td>64-0712</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator bath</td>
			<td>Branson</td>
			<td>1510R-MT</td>
			<td>Bransonic Ultrasonic cleaner. 50-60 Hz. Output: 70W</td>
		</tr>
		<tr>
			<td>Soy PI</td>
			<td>Avanti Polar Lipids</td>
			<td>840044</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop centrifuge</td>
			<td>Eppendorf</td>
			<td>22331</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Lamp</td>
			<td>Spectroline</td>
			<td>ENF-260C</td>
			<td>115 Volts, 60 Hz, 0.20 AMPS</td>
		</tr>
		<tr>
			<td>WhatmanGlass Microfiber Filter Paper</td>
			<td>VWR</td>
			<td>28455-030</td>
			<td>42.5 mm diameter, Grade GF/C</td>
		</tr>
	</tbody>
</table>

reconstitution of septin assembly at membranes to study biophysical properties and functions

大多数细胞可以感知并改变它们的形状，以执行基本的细胞过程。在许多真核生物中，隔膜细胞骨架是协调形状变化（如细胞分裂，极化生长和迁移）的组成部分。Septins是形成细丝的蛋白质，它们组装形成各种高阶结构，并且在许多情况下，存在于质膜的不同区域，最显着的是微米级正曲率区域。监测 体内 隔膜组装过程受到细胞中光学显微镜的局限性以及与膜和细胞骨架元件相互作用的复杂性的阻碍，使得难以量化生命系统中的隔膜蛋白动力学。幸运的是，在过去十年中，在无细胞系统中重建隔膜蛋白细胞骨架以剖析以高空间和时间分辨率控制隔膜蛋白组装的机制方面取得了实质性进展。隔膜蛋白组装的核心步骤包括隔膜杂高分子缔合和解离，聚合成细丝，以及通过细丝之间的相互作用形成高阶结构。在这里，我们提出了三种在不同情况下观察隔膜组装的方法：平面双层，球形支撑和杆支撑。这些方法可用于确定隔膜在不同组装阶段的生物物理参数：作为结合膜的单个八聚体，作为细丝，以及作为细丝的组装。我们将这些参数与曲率采样和优先吸附的测量值相结合，以了解曲率传感如何在各种长度和时间尺度上工作。

在制备每个SLB之后，可以将分隔蛋白或目标蛋白质与所需的支持物一起孵育，并通过TIRFM，共聚焦显微镜或SEM成像。这里显示的结果使用从 大肠杆菌17重组表达和纯化的隔膜蛋白。在平面SLB上使用TIRFM，可以确定灯丝的长度及其柔韧性，测量扩散系数并观察28，29随时间变化的装配。为了收集最高质量的测量结果，首先需要确定双...

Watch this Scientific Journal Video about Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions at JoVE.com

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

无细胞重建一直是了解细胞骨架组装的关键工具，并且在过去十年中的工作已经建立了研究最小系统中隔膜蛋白动力学的方法。这里介绍的是三种在不同膜环境中观察隔膜组装的互补方法：平面双层，球形支撑和棒状支撑。

在膜上重构隔膜组件以研究生物物理性质和功能

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

该协议将指导那些从事膜结合蛋白工作的人确定膜形状如何在蛋白质组装的组织中发挥作用。该技术的主要优点是其在蛋白质类型、膜组成和膜几何形状方面的多功能性。它还允许询问脂质双层如何改变相关蛋白质的性质。为了确定适合实验的膜，我们建议建立一个清单，其中可能包括脂质扩散速率，目标蛋白质的迁移率，以及是否有未破裂的脂质体或融合的脂质体粘在表面。要开始对载玻片进行等离子清洁，请用氧气吹扫等离子清洁器五分钟，以去除管路和腔室中的空气。将干燥的盖玻片载玻片排列到陶瓷支架中。吹扫时，将惰性气体流过微型盖玻片载玻片，以去除灰尘和颗粒。将支架放在等离子体室的背面，使盖玻片与腔室的长边缘平行。以最大功率用氧气运行等离子清洁器15分钟。对于腔室制备，切下 0.2 毫升 PCR 管磨砂部分下方的盖子。用紫外线激活粘合剂涂漆PCR管的边缘，避免进入管内。轻轻地将PCR管胶水放在等离子清洁盖玻片的中心，然后将腔室置于长波长紫外线下五到七分钟以固化粘合剂。要形成双层，请将试剂添加到孔中。轻轻左右摇晃腔室以破坏 SUV，然后在 37 摄氏度下孵育 20 分钟。孵育后，通过移液用150微升SLBB冲洗双层六次以洗去多余的脂质，然后用150微升反应缓冲液洗涤双层六次，然后与隔膜一起孵育。在最后一个洗涤步骤中，除去反应缓冲液，在孔中留下75微升。加入 25 μL 在 SSB 中稀释的隔膜素，并通过 TIRF 显微镜成像。首先，涡旋含有二氧化硅微球的瓶子15秒，然后浴超声处理一分钟，然后再次涡旋15秒以打破任何团簇。将珠子与SLBB和10微升五毫摩尔SUV在低粘附微量离心管中混合。将珠状脂质混合物在室温下在摇床上孵育一小时以防止沉淀。同时，解冻聚乙二醇化的盖玻片并将切好的0.2毫升PCR管粘在其上。孵育后，在指定的RCF下将珠子离心30秒。第一次旋转后，除去50微升上清液，然后加入200微升PRB并通过剧烈移液混合。对于下一轮，去除 200 微升 PRB，并在第二次和第三次旋转后再添加 200 微升，并在第四次旋转后添加 220 微升 PRB。如果使用多个磁珠尺寸进行竞争测定，则通过将等体积的每个磁珠尺寸混合并将29微升该珠混合物与721微升反应缓冲液稀释来制备反应，然后将75微升稀释的珠子和25微升在SSB中稀释的蛋白质加入孔中并混合。如果在稳定状态下测量隔膜素，请将混合物在室温下孵育一小时，然后通过TIRF附近或共聚焦显微镜成像。与绿色隔膜一起孵育的平面SLB的TIRF显微照片显示均匀的蛋白质分布。用清洁不良的玻璃盖玻片制成的SLB在低隔膜区域的双层中显示出孔洞或间隙。如果使用旧脂质体或洗涤不严格，未爆裂和管状脂质体可能会积聚在表面上。在使用罗丹明PE可视化的脂质包被微球的显微照片中，显示出光滑的双层沉积。球形支撑物均匀地被膜覆盖，并且很少有珠簇。过量脂质的洗涤不充分导致膜涂层不均匀，如脂质团块所观察到的那样，处理不当导致双层间隙。磁珠混合不充分导致结块，这对于测量总蛋白质吸收并不理想。使用此处介绍的支持脂质双层，可以执行其他方法，例如荧光寿命成像显微镜，膜质细胞术和高速原子力显微镜，以检查不同的蛋白质 - 蛋白质或蛋白质 - 膜动力学。

Research

JoVE Journal

Biochemistry

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

从多个小鼠组织分离中间丝状蛋白以研究与老化相关的翻译后修饰

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

科研

生物化学

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

固体核磁共振波谱法研究高分子组件的原子尺度结构

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

光谱分析 TRPV1 的纯化与重构

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products

测量天然产品生物调节特性的平面运动分析

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and ...

Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

膜蛋白共翻译插入预成型纳米盘

Cell-free expression systems allow the tailored design of reaction environments to support the functional folding of even complex proteins such as ...

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

用完全纯化的组分重构Msp1提取活性

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...